Presynaptic CK2 promotes synapse organization and stability by targeting Ankyrin2

The precise regulation of synapse maintenance is critical to the development and function of neuronal circuits. Using an in vivo RNAi screen targeting the Drosophila kinome and phosphatome, we identify 11 kinases and phosphatases controlling synapse stability by regulating cytoskeletal, phospholipid, or metabolic signaling. We focus on casein kinase 2 (CK2) and demonstrate that the regulatory (β) and catalytic (α) subunits of CK2 are essential for synapse maintenance. CK2α kinase activity is required in the presynaptic motoneuron, and its interaction with CK2β, mediated cooperatively by two N-terminal residues of CK2α, is essential for CK2 holoenzyme complex stability and function in vivo. Using genetic and biochemical approaches we identify Ankyrin2 as a key presynaptic target of CK2 to maintain synapse stability. In addition, CK2 activity controls the subcellular organization of individual synaptic release sites within the presynaptic nerve terminal. Our study identifies phosphorylation of structural synaptic components as a compelling mechanism to actively control the development and longevity of synaptic connections.     

PASSAGE OF 5-HYDROXYTRYPTAMINE THROUGH THE BLOOD-BRAIN BARRIER, ITS METABOLISM IN THE BRAIN AND ELIMINATION OF 5-HYDROXYINDO-LEACETIC ACID FROM THE BRAIN TISSUE

Abstract— 5-HT was injected intravenously in rats (10 mg/kg) and a marked increase in brain 5-HT and 5-HIAA was observed. For the first 10 min after injection the penetration of 5-HT into the brain and formation of 5-HIAA is evident. After 10 min degradation of exogenous 5-HT and elimination of 5-HIAA are prominent. Metabolism of exogenous 5-HT in the brain is very fast (half-life between 5 and 10 min; completely metabolized in approximately 80 min). The importance of these results in explaining the permeability of blood-brain barrier to 5-HT is discussed. Experiments on brain slices show that 5-HT is more readily metabolized in brain tissue than eliminated into incubation medium. In contrast, 5-HIAA very easily leaves brain tissue.     

The structure and activity of fungal lipases in bile-salt solutions

The changes in the structure and catalytic properties of fungal lipases (Candida rugosa, Rhizomucor miehei, Mucor javanicus) were investigated in micellar solutions of bile salts that differ in their hydrophilic–lypophilic balance and reaction medium properties. The methods of circular dichroism and tryptophan fluorescence were applied to estimate the changes in peptide structure within complexes with bile-salt micelles. Bile salts do not exert a significant influence on the structure of the enzymes under study: in the Rh. miehei and M. javanicus lipases the α-helix content was slightly decreased; an influence of the bile salts on the C. rugosa structure was not revealed. Despite negligible structural modifications in the enzymes, a considerable change in their catalytic properties, namely an abrupt decrease in catalytic effectiveness was observed in bile-salt solutions. Substrate–bile salt micelle complex formation was demonstrated by the NMR self-diffusion method. A model of the regulation of fungal lipase activity was proposed.     

Comparison of (13)C(alpha)H and (15)NH backbone dynamics in protein GB1

This study presents a site-resolved experimental view of backbone C(alpha)H and NH internal motions in the 56-residue immunoglobulin-binding domain of streptococcal protein G, GB1. Using (13)C(alpha)H and (15)NH NMR relaxation data [T(1), T(2), and NOE] acquired at three resonance frequencies ((1)H frequencies of 500, 600, and 800 MHz), spectral density functions were calculated as F(omega) = 2omegaJ(omega) to provide a model-independent way to visualize and analyze internal motional correlation time distributions for backbone groups in GB1. Line broadening in F(omega) curves indicates the presence of nanosecond time scale internal motions (0.8 to 5 nsec) for all C(alpha)H and NH groups. Deconvolution of F(omega) curves effectively separates overall tumbling and internal motional correlation time distributions to yield more accurate order parameters than determined by using standard model free approaches. Compared to NH groups, C(alpha)H internal motions are more broadly distributed on the nanosecond time scale, and larger C(alpha)H order parameters are related to correlated bond rotations for C(alpha)H fluctuations. Motional parameters for NH groups are more structurally correlated, with NH order parameters, for example, being larger for residues in more structured regions of beta-sheet and helix and generally smaller for residues in the loop and turns. This is most likely related to the observation that NH order parameters are correlated to hydrogen bonding. This study contributes to the general understanding of protein dynamics and exemplifies an alternative and easier way to analyze NMR relaxation data.     

Heat capacities and a snapshot of the energy landscape in protein GB1 from the pre-denaturation temperature dependence of backbone NH nanosecond fluctuations

Protein stability is usually characterized calorimetrically by a melting temperature and related thermodynamic parameters. Despite its importance, the microscopic origin of the melting transition and the relationship between thermodynamic stability and dynamics remains a mystery. Here, NMR relaxation parameters were acquired for backbone 15NH groups of the 56 residue immunoglobulin-binding domain of streptococcal protein G over a pre-denaturation temperature range of 5-50 degrees C. Relaxation data were analyzed using three methods: the standard three-Lorentzian model free approach; the F(omega)=2omegaJ(omega) spectral density approach that yields motional correlation time distributions, and a new approach that determines frequency-dependent order parameters. Regardless of the method of analysis, the temperature dependence of internal motional correlation times and order parameters is essentially the same. Nanosecond time-scale internal motions are found for all NHs in the protein, and their temperature dependence yields activation energies ranging up to about 33kJ/mol residue. NH motional barrier heights are structurally correlated, with the largest energy barriers being found for residues in the most "rigid" segments of the fold: beta-strands 1 and 4 and the alpha-helix. Trends in this landscape also parallel the free energy of folding-unfolding derived from hydrogen-deuterium (H-D) exchange measurements, indicating that the energetics for internal motions occurring on the nanosecond time-scale mirror those occurring on the much slower time-scale of H-D exchange. Residual heat capacities, derived from the temperature dependence of order parameters, range from near zero to near 100J/mol K residue and correlate with this energy landscape. These results provide a unique picture of this protein's energy landscape and a relationship between thermodynamic stability and dynamics that suggests thermosensitive regions in the fold that could initiate the melting process.     

Internal motional amplitudes and correlated bond rotations in an alpha-helical peptide derived from 13C and 15N NMR relaxation

Peptide GFSKAELAKARAAKRGGY folds in an alpha-helical conformation that is stabilized by formation of a hydrophobic staple motif and an N-terminal capping box (Munoz V. Blanco FJ, Serrano L, 1995, Struct Biol 2:380-385). To investigate backbone and side-chain internal motions within the helix and hydrophobic staple, residues F2, A5, L7, A8, and A10 were selectively 13C- and 15N-enriched and NMR relaxation experiments were performed in water and in water/trifluoroethanol (TFE) solution at four Larmor frequencies (62.5, 125, 150, and 200 MHz for 13C). Relaxation data were analyzed using the model free approach and an anisotropic diffusion model. In water, angular variances of motional vectors range from 10 to 20 degrees and backbone phi,psi bond rotations for helix residues A5, L7, A8, and A10 are correlated indicating the presence of Calpha-H, Calpha-Cbeta, and N-H rocking-type motions along the helix dipole axis. L7 side-chain CbetaH2 and CgammaH motions are also correlated and as motionally restricted as backbone CalphaH, suggesting considerable steric hindrance with neighboring groups. In TFE which stabilizes the fold, internal motional amplitudes are attenuated and rotational correlations are increased. For the side chain of hydrophobic staple residue F2, wobbling-in-a-cone type motions dominate in water, whereas in TFE, the Cbeta-Cgamma bond and phenyl ring fluctuate more simply about the Calpha-Cbeta bond. These data support the Daragan-Mayo model of correlated bond rotations (Daragan VA, Mayo KH, 1996, J Phys Chem 100:8378-8388) and contribute to a general understanding of internal motions in peptides and proteins.     

A Method for Chronic Registration of Brain Cortical Electrical Activity in Rats

Journal of Evolutionary Biochemistry and Physiology - Electrocorticogram registration and analysis (electrocorticography, ECoG) is widely used in small-animal biomedical research. To date, a...     

2′-OH of mRNA are critical for the binding of its codons at the 40S ribosomal P site but not at the mRNA entry site

The roles of 2′-OH groups in the binding of mRNA to human ribosomes were studied using site-directed cross-linking. We found that both mRNA and mDNA analogues bearing a cross-linker can modify ribosomal proteins (rps) S3e and S2e at the mRNA entry site independently on tRNA presence, but only mRNA analogues were capable of a tRNA^Phe-dependent binding to human ribosomes and cross-linking to rpS26e in the mRNA binding centre. Thus, 2′-OH groups of mRNA are unimportant for binding at the entry site but they are crucial for codon-anticodon interactions at the P site, implying the existence of mRNA-ribosome contacts that do not occur in bacteria.     

Transventricular and transpial absorption of cerebrospinal fluid into cerebral microvessels

It is generally accepted that volume of cerebrospinal fluid (CSF) is secreted in brain ventricles and flows to subarachnoid space to be absorbed into dural venous sinuses or/and into lymphatics via perineural sheats of cranial nerves. Since 99% of CSF volume is water, in experiments on cats 3H-water was slowly infused into lateral ventricle and found that it does not flow to subarachnoid space but that it is rapidly absorbed transventricularly into periventricular capillaries. When 3H-water was infused in cortical subarachnoid space, it was absorbed locally into cerebral capillaries via pia mater. On the contrary, when macromolecule 3H-inulin is applied in CSF it is very slowly eliminated in bloodstream, and, with time, is carried by systolic-diastolic pulsations and mixing of CSF bidirectionally along CSF system. Thus, CSF volume (water) is absorbed rapidly into adjacent cerebral capillaries while inulin is distributed bidirectionally due to its long residence time in CSF Previously, the macromolecules have been used to study CSF volume hydrodynamics and with this misconception of CSF physiology arose.